Antibodies are powerful and invaluable tools in diagnostics and therapeutics. In accordance with the growing demand of highly specific antibodies, a number of different strategies have been developed over time in order to allow a reliable production of antibodies in sufficient amounts (see, e.g., J. S. Haurum, Drug Discovery Today, 2006. 11(13-14), 655-660). In this context, the discovery of the hybridoma technology in 1975 represents a significant breakthrough as mouse hybridomas were the first reliable source of monoclonal antibodies.
In theory, monoclonal antibodies can be developed against any target of choice. The provision of the target of choice is absolute essential. Even in the case of genetic immunization the antigen is required for screening and confirmation purposes. Unfortunately, in some cases provision of the antigen might become a problem if neither antigen isolation nor production in cell-based assays is successful.
Usually, the antigen belongs to the class of proteins and peptides, but principally also other substance classes can be used as immunogen as for example polysaccharides, lipids and polynucleotides. In most cases, antibodies shall be generated against the native antigen. If the antigen is produced by recombinant DNA technology, it is very important to compare the properties of the resulting recombinant protein or peptide with its native counterpart since differences in the protein structure may lead to differences in the immunogenicity. Proteins and peptides are highly interesting targets for antibody production. Peptide synthesis represents an alternative for cell-based expression given the fact that the immunogenic epitope is linear and not dependent on the tertiary structure of the intact protein. On the other hand, peptide synthesis has its limitations for proteins and peptides with posttranslational modifications (PTMs) which are part of the three-dimensional epitope. Furthermore, usage of peptides and peptide mixtures as antigen may induce the generation of cross-reactive antibodies.
Membrane proteins account for nearly one third of all sequenced genomes. In contrast to their crucial involvement in cellular processes such as signaling, metabolism, transport and recognition, little information is available about membrane protein structures and functionality. The deregulation of their biological activity in vivo can lead to severe diseases, making them highly interesting pharmacological targets. This development caused an increase in the demand of membrane protein specific antibodies.
The generation of specific antibodies against membrane proteins is often limited by the inability to produce or isolate a certain target membrane protein. Overexpression of membrane proteins in vivo is often hampered by protein misfolding, insolubility, aggregation, low productions yields and cell cytotoxicity. However, cell-free expression of membrane proteins can overcome these obstacles.
Over the last decade, cell-free protein synthesis has become a valuable tool for the production of different protein classes including membrane proteins, cytosolic or even toxic proteins (see, e.g. Katzen et al., Trends Biotechnol., 2005. 23(3), 150-156). The basis of an efficient cell-free expression system is a translationally active cell extract which contains the essential components for translation such as ribosomes, translation factors and enzymes. Furthermore, the cell lysate is supplemented with amino acids, ATP and GTP and an energy regenerating system (e.g. creatine-phosphate/creatine-kinase energy-regenerating system). Synthesis of the target protein is initiated by addition of an appropriate template either in form of DNA or mRNA. Until now, many different types of functionally active target proteins have been expressed in prokaryotic and eukaryotic cell-free systems. In comparison to prokaryotic cell extracts eukaryotic cell lysates offer several advantages in the expression of complex proteins which require posttranslational modifications.
Nevertheless, the use of the proteins synthesized by means of prokaryotic and eukaryotic cell lysates for the production of specific antibodies according to the conventional methods of the prior art still involves additional laborious purification and/or isolation steps in order to provide the desired target protein in a suitable form for use as an immunogenic substance.
In view of the drawbacks of the prior art, the main object of the present invention was to provide improved methods and means for producing polyclonal antibodies against a specific target protein.
This objective has been achieved by providing the novel methods for producing polyclonal antibodies against a specific target protein using an antigenic composition comprising protein-containing membrane vesicles according to the invention.